Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007General ConclusionThis research proposal focuses on the various regulative influences the mitochondria have on thecell cycle and cell cycle progression, and vice versa. The goal of this research program, consisting of fourprojects, is to establish a more complete picture of the mechanisms through which the cell cycle and themitochondria correspond with one another. The links between the cell cycle and the mitochondria areimportant to be established as the mechanisms regulate to some extend the two very important cellularprocesses of cell cycle progression and mitochondrial activity. Next to the gaining of knowledge that willbe achieved throughout the conductance of the proposed research, better understanding of severaldiseases, such as cancer and Charcot Marie tooth may be developed.In the first project, the existence of a metabolic cycle was discussed. ROS fluctuation throughout the cellcycle is proposed to be studied. In addition, the relative contribution of mitochondria to ROS productionshould be investigated in comparison to other known ROS producers. Lastly, the influence of ROS on theG2/M phase transition should be explored in order to establish a clear link on how ROS levels regulate cellcycle progression. Through performing this research, a strong interaction between the cell cycle andmitochondria through ROS might be indicated.In the second part of this proposal, focus was placed on the energy checkpoint in G1 phase. Thepathways through which high levels of activated AMPK can induce cell cycle arrest should be explored. Alink between energy-producing mechanism and cell cycle arrest is aimed to be better understood, and adirect link between AMPK and energy-producing mechanisms is proposed. Lastly, it will be investigatedwhether, and how, mitochondrial morphology changes after the cell goes into cell cycle arrest. Throughthe accomplishment of this research proposal, the role and functionality of AMPK in the regulativeinteraction between energy status in the cell and cell cycle arrest will be better determined.Consecutively, a research that is mainly focused on the role of the cell cycle for mitochondrialbiogenesis was proposed. This research is necessary to improve the perception of the dependence ofmitochondrial biogenesis on cell cycle progression. The role of nuclear respiration factor (NRF) is ofparticular interest as it is a factor which is capable of activating the transcription of various mitochondrialcomponents. The main aim of this research is to investigate the dynamics of mitochondrial biogenesisduring the cell cycle and to understand how induction of mitochondrial biogenesis by different factors maybe coordinated.Lastly, the regulative interaction between mitochondria and cell cycle progression is aimed to beexplained through the activities of mitofusin2. The fluctuations of Mfn2 levels throughout the cell cycle areinvestigated and a possible mechanism of oxidative phosphorylation maintenance with the aid Mfn2 ishypothesized. A mechanism of Mfn2 cleavage is proposed. Through the latter, the generation of both themitochondrial and the cytosolic isoforms with their opposing roles may be explained. Furthermore, itinvestigates the ability of cytosolic Mfn2 to inhibit Ras. Lastly, the influence of cyclins on Mfn2 levels andactivity are assessed to round up the circle of regulative interaction between the cell cycle andmitochondria through Mfn2.To conclude, the four projects share in common much more than merely the general theme ofresearch. Both project one and two shed a focus on metabolic activities, which is also a common aspectwith project four. Even though throughout the different projects several pathways to cell cycle arrest areexplored independently, in practice of reality they might all be linked to a general pathway of cell cyclearrest. Project three focuses on the nuclear encoding of mitochondrial proteins. Mfn2, discussed in projectfour might be influenced by NRF. Since the four topics are linked by various means, a complete picturewill not be provided throughout one individual project. Rather, throughout the performance of all fourproposals, a more lucid picture will be created, and a broader platform of background information forfuture research, will be provided.SCI 332 Advanced Molecular Cell Biology Research Proposal 94
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Appendix A[35] S Incorporation[35S]-cysteine and [35S]-methionine can be used to label cells in pulse chase analyses. Metaboliclabeling of this kind is made possible by the low-energy beta-emitting radioisotopes ([35S]-cysteine and[35S]-methionine) and are often used to track in vivo biosynthesis, maturation or degradation of proteins.AICAR5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) is an AMP-activated protein kinase (AMPK)activator. Rat hepatocyte incubation with AICAR results in an accumulation of a monophosphorylatedderivative called 5-aminoimidaz-ole-4-carboxamide ribonucleoside (ZMP) inside the cell. ZMP has amimicking action of both activating effects of AMP on AMPK and promotes phosphorylation by AMPKkinase.This technique is particularly advantageous because unlike other methods for activating AMPK inintact cells, AICAR does not disturb the cellular contents of ATP, ADP or AMP. AICIAR is a usefultechnique for identifying new target pathways and regulative processes controlled by the protein kinasecascade.Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Microplate Fluorometer)The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit is a single step, fluorometric assay with highsensitivity. The assay is able to detect quantities as small as 10 picomoles of hydrogen peroxide (H 2 O 2 )or 10 µU/mL of horseradish peroxidase activated in a 100 µL volume of assay.The Amplex Red reagent is one of the most stable and sensitive fluorogenic substrates availablefor horseradish peroxidase and allows for a variety of fluorogenic and chromogenic assays for enzymesproducing hydrogen peroxide. Assays using this reagent are capable of the extremely sensitivequantification of various analytes such as glucose, galactose, cholesterol and many more as well ashydrogen peroxide.This kit provides an easy, sensitive way to detect H 2 O 2 or the horseradish peroxidase bymeasuring fluorescence with a microplate fluorometer. The substrate in this kit enables detection of activeperoxidases and H 2 O 2 release from cells as well as detection of H 2 O 2 production from enzyme-coupledreactions.BrdU & DAPI Cell CycleThe synthetic nucleoside, Bromodeozyuridine (5-bromo-2-deoxyuridine, BrdU) is commonly applied fordetection of proliferating cells in living tissues. Incorporation of BrdU into new synthesized DNA ofreplicating cells during the S phase substitutes the role of thymidine during DNA replication. The techniqueapplies specific BrdU antibodies, which allow the detection of the chemical by immunohistochemistry, thisthen confirms whether cells are actively replicating their DNA.4’,6-diamidino-2-phenylindole (DAPI) is a fluorescent stain capable of strong binding to DNA. TheDAPI stained is used particularly in fluorescence microscopy, where DAPI is excited with ultraviolet light.DAPI is able to pass through an intact cell membrane and is therefore a useful stain in detection of DNA inboth live and fixed cells.cDNAStrands of cDNA or copy DNA can be either single stranded or double stranded. In vitro synthesis ofcDNA from an mRNA template can be achieved by using reverse transcriptase, which produces singlestranded cDNA. This process is referred to as reverse transcription or first strand cDNA synthesis.Microarray experiments require double stranded cDNA, which can be produced through anotherround of DNA synthesis after the first strand cDNA synthesis. Conversion of mRNA to cDNA is usedprimarily in template mRNA analysis because DNA is more stable than RNA. cDNA conversion alsoallows use in RT-PCR, as a probe for analysis of expression of mRNA sequences.SCI 332 Advanced Molecular Cell Biology Research Proposal 95
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